The regulatory region of the L-arabinose operon: a physical, genetic and physiological study.

A fine-structure physical and genetic map was constructed of a 1000 base-pair region of the l-arabinose operon of Escherichia coli. This region consists of the ara regulatory sequences contained between the araC and araB genes and portions of these flanking genes. Point mutations, Mu phage insertions, and bacterial deletions as well as arabinose-induced and basal enzyme levels in the strains were used in constructing a genetic map of the region. These ordered positions were then located more accurately by mapping the point mutations against physically located endpoints of deletions isolated on the two non-defective transducing phage λparaB114 and λparaC116. Phage possessing deletions ending in the arabinose regulatory region were isolated from indicating-plates on which deletions removing none, part, or all of either the araC or araB genes carried on the phage could be distinguished. Phage stocks were enriched for such deletions prior to plating by treatment with chelating agents and heat (Parkinson &; Huskey, 1971). Deletions into the ara region on either phage shorten the ara DNA homology region formed from heteroduplexes between λparaB114 and λparaB116. Therefore the physical locations of these deletion endpoints were determined by electron microscopy of the appropriate heteroduplexes and/or by gel electrophoresis of the central duplex following S₁ nuclease digestion (Lis &; Schleif, 1975b). 18 of the 32 deletions isolated and mapped in this region were measured physically. The space between araC and araB, containing the regulatory elements of the operon, is estimated to be about 300 base-pairs.     

Hotspots for generalized recombination in the Escherichia coli chromosome.

A naturally occurring hotspot for Rec recombination of Escherichia coli was located in the biotin operon. The phenotypes of the bio hotspot as observed in λbio transducing phage were identical to those of Chi mutations in phage λ. In addition to recA⁺ function, the site-specific stimulation of recombination required recB⁺ function. The stimulation took place when the hotspot was present in only one parent of the cross and even when present opposite a region of heterology.The demonstration of a Chi element in E. coli provoked us to measure the density of Chi elements on the chromosome. E. coli DNA sampled in λ transducing phage (either obtained by induction of secondary site lysogens or made in vitro from EcoRI cleavage fragments) showed one hotspot per 5 to 15 × 10³ bases. The high density and the fact that Chi stimulation of recombination can span the inter-Chi distance suggest that Chi might be important in Rec recombination in the absence of λ.     

Gene N regulator function of phage λimm21: Evidence that a site of N action differs from a site of N recognition

We report the isolation and characterization of a new mutation in the hybrid phage λimm21. Both genetic and physiological studies demonstrate that this new mutation, N_21?1, is similar to N mutations of phage λ. As in the case of the N gene of λ (Ni_λ), the N_21?1 mutation maps immediately to the left of the cI gene and has a pleiotropic effect on the expression of phage functions. Although these studies strongly suggest that phage 21 has an N function, they do not definitely locate the N_21?1 mutation within the N₂₁ structural gene.Reported here are studies demonstrating that N₂₁ acts in trans, similar to N_λ, to stimulate the expression of phage functions. N products show an immunity specificity; N₂₁ being only active on phage carrying the immunity region of phage 21, while the n_λ is only active on phage carrying the immunity region of λ or phage 434. However, one site of action for N_λ can be rescued from phage 21. We propose that the specificity of an N function is determined by its sites of recognition and that these sites may be different from the sites of N action.     

Polarity suppressors increase expression of the wild-type tryptophan operon of Escherichia coli

Three new polarity suppressors, selected to relieve the polar effect of nonsense mutations in the tryptophan (trp) and lactose (lac) operons of Escherichia coli, increase expression distal to nonsense mutations in both operons to a greater extent than suA. These suppressors relieve the polarity created by amber, ochre and frameshift mutations with equal efficiency.Two of the three polarity suppressors elevate enzyme synthesis in the wildtype trp operon two- and fivefold, respectively. The increase in enzyme levels is in each case correlated with increased levels and rates of synthesis of structural gene trp messenger RNA. Since expression of all genes is elevated, these findings suggest the existence of a site early in the wild-type trp operon that affects the extent of operon expression. We located the site affected by these two polarity suppressors between the operator and the first structural gene, trpE. Although the third polarity suppressor also relieves mutational polarity efficiently, it has no detectable effect on expression of the wild-type trp operon.     

Polarity induced by chloramphenicol and relief by suA

The suA allele, known to relieve polarity in Escherichia coli, also relieves a unique polar effect on E. coli tryptophan operon messenger RNA produced by chloramphenicol.     

Regulation of mRNA Utilization and Degradation by Amino-acid Starvation

Amino-acid starvation of a stringent strain of E. coli relieves the polar effect on distal messenger RNA imposed by a nonsense mutation. There is no relief of polarity starvation of a relaxed strain. In fact, starvation of a relaxed (but not of a stringent) strain by itself causes an artificial polar effect on distal mRNA. These findings are consistent with a mechanism for polarity based on mRNA degradation.